In article <1993Aug24.013048.29384 at gserv1.dl.ac.uk> Nelson Salazar,
N.SALAZAR at uk.ac.lshtm writes:
>Hi everybody,
> I would also appreciate very much if someone could
>help us out with the sequencing of PCR products without having to
>clone them. I have used the Pharmacia kit, which seems quite simple,
>but I failed the first time. Now I am preparing more template DNA
>(ssDNA) and I hope that this time it will work. However, if someone
>has used this approach or has a better one, that would help us quite
>a bit.
>>Thanks very much and I look forward for your responses.
Here is my e-mail reply to the original poster of this thread:
I have had fair success in directly sequencing RT-PCR products. The
limitations are quality and quantity of the PCR product. I have tried
two ways of direct sequencing of PCR products, and there is still one
other I have not tried yet.
1) 32-P labelling of the sequencing primer: I have not tried yet.
2) Doing the PCR with one primer 5' phosphorylated by T4 polynucleotide
kinase, followed by lambda phage exonuclease digestion of the PCR product
to reduce it to single stranded form. I tried this and had problems, but
may have given up too quickly. See Higuchi & Ochman Nucl. Acids Res.
17:5865 (1989) and Lee et al. J Virol Methods 37:275-288 (1992) for
details. The thread you remember was dominated by someone who had good
success using this method, but lousy results using asymmetric PCR
products.
3) Asymmetric PCR, in which the two primers are present in 50:1 molar
ratio. This is the one that has given me the most success. Currently, I
run out the products of the RT-PCR on low-melt agarose and cut out the
band(s) of interest. After purifying by phenol, then phenol:chloroform
and lastly chloroform extraction and EtOH precipitation I do a regular
PCR and again isolate the product from low-melt agarose. If this
reaction looks clean, I run the asymmetric PCR without anything more than
EtOH precipitation from 2.5M ammonium acetate and a 70% EtOH rinse.
Dissolve the product in 20ul TE. The Sequenase 2.0 kit is used for
sequencing with a modification to the annealing conditions: 2ul
Sequenase buffer, 1ul primer and 7ul PCR product are heated for 5 min. at
>95 deg C, then quick chilled on ice. (Check the volume; generally 1-2ul
water is needed because of evaporation at 95 deg.) The labeling and
termination reactions are run as soon as possible. I have had good
results with this method, although sometimes the PCR product is
heterogeneous and the sequence is hard to read.
Good luck,
Jim Owens